We present evidence for the presence of cryptic kininogen-activated cysteine protease in Trypanosoma cruzi, the causative agent of Chagas' disease. T. cruzi lysates exhibited major bands of proteolytic (cruzipain) activity in the 45-55 kDa molecular mass range in gelatin-containing SDS-PAGE gels. Addition of kininogen (a cystatin-like protease inhibitor) to the lysates before electrophoresis paradoxically resulted in the appearance of additional bands of proteolytic activity in the 160-190 kDa molecular mass range. Preincubation at low temperatures (-20C optimum) greatly enhanced the proteolytic activity suggesting that a metastable complex forms between kininogen and a cryptic 30 kDa cysteine protease from T. cruzi and that this activity participates in the proteases activation.